Development 127, 769-778 (2000) 769 Printed in Great Britain © The Company of Biologists Limited 2000 DEV7727

Formation and specification of distal leg segments in by dual Bar genes, BarH1 and BarH2

Tetsuya Kojima*, Makoto Sato and Kaoru Saigo Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan *Address for correspondence (e-mail: [email protected])

Accepted 29 November 1999; published on WWW 26 January 2000

SUMMARY

Here, we show that BarH1 and BarH2, a pair of Bar functions, early antagonistic interactions with a pretarsus- homeobox genes, play essential roles in the formation and specific homeobox gene, aristaless, and the subsequent specification of the distal leg segments of Drosophila. In induction of Fas II expression in pretarsus cells abutting early third instar, juxtaposition of Bar-positive and Bar- tarsal segment 5. Bar homeobox genes are also required for negative tissues causes central folding that may separate specification of distal tarsi. Bar expression requires Distal- future tarsal segments 2 from 3, while juxtaposition of less but not dachshund, while early circular dachshund tissues differentially expressing Bar homeobox genes at expression is delimited interiorly by BarH1 and BarH2. later stages gives rise to segmental boundaries of distal tarsi including the tarsus/pretarsus boundary. Tarsus/pretarsus Key words: Leg, Homeobox gene, Segmentation, BarH1, BarH2, Fas boundary formation requires at least two different Bar II, aristaless, Distal-less, dachshund, Drosophila

INTRODUCTION of the concentric domains occurs in multiple phases. The coxopodite and telopodite regions are initially established by The formation of compartments or domains specified by the the expression of Dll and a homeobox gene, homothorax (hth). region-specific expression of transcription factors may be In coxopodite, the proximalmost region constituting future essential for the of and (e.g. for body wall and proximal segments, Dpp and Wg signaling are Drosophila, see Azpiazu et al., 1996; Lawrence and Struhl, blocked by concerted function of Hth and nuclear localized 1996; Sato et al., 1999). Drosophila leg development may Extradenticle (Exd), another homeodomain protein (Abu- provide a good system for studying region-specific expression Shaar and Mann, 1998; Gonzalez-Crespo and Morata, 1996; and compartment formation, since legs are simple in structure Wu and Cohen, 1999). In contrast, in the telopodite, Dpp and and their formation encompasses various developmental Wg signaling are active and required for proximodistal axis processes. generation. Subsequently, a dac expression domain appears Drosophila legs comprise the segmental units, from between hth and Dll domains to subdivide the telopodite region proximal to distal, coxa, trochanter, femur, tibia, tarsal further. One more intermediate region, which expresses both segments 1-5 and pretarsus. Leg formation occurs through dac and Dll, then arises by the early third instar. concentric folding and subsequent segmentation of Downstream of Dll, several genes participate in distal tarsal monolayered epithelia of leg discs invaginated from the formation. A homeobox gene aristaless (al), expressed in the epidermis during embryogenesis (Cohen, 1993). According to most distal tip of leg discs, is required for the pretarsus Lecuit and Cohen (1997), decapentaplegic (dpp) and wingless structures (Campbell et al., 1993; Schneitz et al., 1993; (wg) expressed dorsally and ventrally, respectively, along the Campbell and Tomlinson, 1998). spineless (ss), which encodes anteroposterior compartment boundary (AP boundary) are a bHLH-PAS protein homologous to mammalian dioxin essential for the concentric expression of Distal-less (Dll) and , is expressed transiently in a future tarsus region and dachshund (dac) in leg discs. Dll, encoding a homeodomain its absence results in deletion of distal tarsal segments 2-4 protein, is expressed in the distal region of leg discs and is (Duncan et al., 1998). bric á brac (bab), encoding a nuclear required for the development of all distal structures other than factor having BTB domain, is expressed downstream of ss the coxa (Cohen et al., 1989; Diaz-Benjumea et al., 1994). dac, (Duncan et al., 1998) and is essential for the segmentation and encoding a novel nuclear protein, is expressed in the middle specification of tarsal segments 2-4 (Godt et al., 1993). leg region and is essential for the formation of intermediate Here, we show that functionally redundant homeobox genes portions of legs (Mardon et al., 1994). at the Bar locus, BarH1 and BarH2 (Kojima et al., 1991; Abu-Shaar and Mann (1998) suggested that the generation Higashijima et al., 1992a), serve as essential regulators in 770 T. Kojima, M. Sato and K. Saigo numerous aspects of distal tarsus development. BarH1 and anti-Ap (Lundgren et al., 1995), mouse anti-Fas II (Lin et al., 1994), BarH2 expression requires Dll. Juxtaposition of Bar-positive mouse anti-Dac (Mardon et al., 1994), rat anti-Al (Campbell et al., and Bar-negative tissues induces initiation of central folding. 1993), mouse anti-Dll (Diaz-Benjumea et al., 1994), rabbit anti-β-gal Antagonistic interactions between Bar and Al or other (Cappell), mouse anti-β-gal (Promega) and Rhodamine-phalloidin pretarsus factors establish the pretarsus/tarsal-segment-5 (Molecular Probe). boundary. Bar is also required for the segmentation and specification of tarsal segments 3-5. RESULTS

MATERIALS AND METHODS Concentric expression of Bar homeobox genes and some distal tarsus segment markers in third instar Fly stocks larvae Fly strains used are: Canton S (wild-type), Df(1)B263-20 (Sato et al., During development of Drosophila legs, the disc epithelium 1999), Df(1)BH2 (Higashijima et al., 1992b), In(1)BM2, In(1)pdf, BSY folds concentrically; genes expressed circularly in the leg disc (Lindsley and Zimm, 1992), In(1)wVC (Hotta and Benzer, 1976), SA1 3 1 just before the onset of folding may thus be essential for leg Dll (Gorfinkiel et al., 1997), dac (Mardon et al., 1994) and al , . BarH1 and BarH2, may belong to such a class al2 (Lindsley and Zimm, 1992). al1 and al2 are used in combination with Df(2L)al (Lindsley and Zimm, 1992). In al1/Df(2L)al and of genes. Staining for BarH1, BarH2 and Bar-lacZ (Fig. 1E,F) al2/Df(2L)al flies, aristae and claws were almost completely abolished indicated that BarH1 and BarH2 are coexpressed circularly in but other pretarsus structures were not significantly affected. en-lacZ all three types of third-instar leg discs. Since BarH1 and BarH2 (ryXho25; Hama et al., 1990), neu-lacZ (A101; Bellen et al., 1989), are functionally redundant to each other (see below), they are Bar-lacZ (P058; FlyView: http://pbio07.uni-muenster.de/) and ta5- hereafter referred to as Bar collectively. lacZ (BM25; T. Michiue and K. S., unpublished data). B55 was Indications of circular Bar expression (Bar ring) were first isolated by imprecise excision of P-element of P058. ptc-GAL4 evident at 76 hours AEL (after egg laying) at 25¡C and became (559.1; Brand and Perrimon, 1993), blk-GAL4 (40C.6; Morimura et more evident at 80 hours AEL (Fig. 1A,B). The formation of al., 1996), ap-GAL4 (md554; Calleja et al., 1996), UAS-BarH1, UAS- the central fold, from which distalmost leg segments are BarH2 (Sato et al., 1999) and UAS-al. UAS-al was generated by generated (see below), began at 84 hours AEL as a circular inserting al cDNA (Campbell et al., 1993) into pUASV (Sato et al., 1999). For FRT/FLP mosaic analyses, hsFLP, FRT18A, FRT40A and indentation along the periphery of the Bar ring, starting FRT42D (Xu and Rubin, 1993) were used. All other mutations and dorsally and completing ventrally by 88 hours AEL (Fig. balancer chromosomes were described in Lindsley and Zimm (1992). 1C,D,G-G′′). As the indentation became deeper in mid third instar, graded Bar expression gradually became apparent along Gynandromorph mosaic analysis the proximodistal axis (Fig. 1H-H′′). In the central fold of a Ten out of twenty eight mosaic legs from 83 flies of the genotype 112 hours AEL leg disc, future tarsal segment 3, lacking Bar Df(1)B263-20, y/In(1)wVC, are entirely Bar−. To isolate Bar− discs, ′′ − expression, became clearly identifiable (Fig. 1I-I ). At 5 hours larvae having mouth hooks and denticle belts mosaic for y were APF (after puparium formation), Bar expression was closely dissected. Among leg discs from eighteen such larvae, six were judged − related to lines of demarcation of tarsal segments (Fig. 1J-L,O). as Bar because of the absence of anti-BarH1 antibody-staining Bar expression was strongest in tarsal segment 5, clearly signals. evident in tarsal segment 4 and not detected in tarsal segment FRT/FLP mosaic analysis 3 and the pretarsus except for future claw regions. Bar−, Dll− and dac− clones, respectively, were generated in larvae ta5-lacZ is a lacZ reporter driven by a tarsal-segment-5- whose genotypes are Df(1)B263-20, y FRT18A/FRT18A; hsFLP, Sb/+, specific Bar (Fig. 1J-M), while ap is a LIM- y w hsFLP; FRT42D DllSA1/FRT42D πM45F and y w hsFLP; dac3 homeobox gene expressed in tarsal segment 4 (Fig. 1M-P; FRT40A/πM36F FRT40A. To examine the ability of BSY to rescue Cohen et al., 1992). Fig. 1Q shows that these markers begin to Df(1)B263-20 mosaic , clones were generated in larvae of be expressed as adjacent circles within the Bar ring during the the genotype Df(1)B263-20, y FRT18A/FRT18A/BSY; hsFLP, Sb/+. In central fold formation, indicating that tarsal segments 4 and 5 all cases, clones were induced by a 90 minute heat shock at 37¡C are derivatives of the early Bar compartment. Because of the during late first-second instar. absence of a suitable molecular marker, we could not Ectopic expression of BarH1, BarH2 and al determine whether future tarsal segment 3 is included in the Several independent UAS-Bar and UAS-al lines were used to early Bar ring. However, since genetic analysis (see below) misexpress Bar and al. UAS-BarH1M6 and UAS-BarH2F9 driven by shows that Bar function in tarsal segments 3-5 is essential for ptc-GAL4 gave leg phenotypes similar to those of blk-GAL4-driven segmentation of distal tarsus, we tentatively conclude that UAS-BarH1M12, UAS-BarH1M13, UAS-BarH2F11 or UAS-BarH2M9. tarsal segments 3-5 are derivatives of the early Bar ring and In combination with ap-GAL4, UAS-BarH1M13, UAS-BarH2M9 and distal tarsus separation starts just prior to the completion of the UAS-BarH2F11 gave leg phenotypes similar to one another. Five central fold formation at early third instar. independent lines of UAS-al showed little leg defects when driven by From late third instar stages onwards, Bar was also ptc-GAL4 or blk-GAL4. expressed in putative claw cells (insets of Fig. 1E,F). Staining Immunohistochemistry for BarH1 and en-lacZ (Fig. 1R; Hama et al., 1990) showed Antibody staining was carried out according to Sato et al. (1999). For the anterior edge of the posterior claw region coincides with confocal microscopy, Cy3-conjugated secondary antibody (Jackson that of the posterior compartment, suggesting that the center of Immune Research) or biotinilated secondary antibody (Vector) the Bar ring is situated in the anterior compartment, between followed by avidin-FITC (Promega) were used. Antibodies used were: the paired claw regions. During late third instar, Bar-negative rabbit anti-BarH1, rabbit anti-BarH2 (Higashijima et al., 1992b), rat patches in future tarsal segment 5 became detectable (Fig. Leg segmentation by Bar homeobox genes 771

Fig. 1. Bar expression in leg discs. cf, central fold; mf, middle fold; pf, peripheral fold; pt, pretarsus; 1-5, tarsal segments 1-5. (A-D) Early (A-C) and mid (D) third instar larval leg discs stained for BarH1. (A) At 76 hours AEL. Note that strong Bar expression occurs in Keilin’s organ cells (arrowheads). (B) Early Bar ring at 80 hours AEL. (C) An 84 hours AEL disc, in which cf is being formed dorsally just outside the Bar ring. mf is not yet formed. (E,F) Late third instar discs stained for Bar-lacZ (red) and BarH1 (E; green) or BarH2 (F; green). BarH1 and BarH2 are coexpressed in the Bar ring region. Arrows, Bar- negative patches in tarsal segment 5. Arrowheads, expression in claw regions. Although BarH1 expression in claw regions is much weaker than BarH2 expression, BarH1/BarH2 coexpression was clearly demonstrated by amplifying BarH1 signals (see the insets). (G-I) Sagittal sections of early (G), mid (H) and late (I) third instar leg discs. Confocal and Nomarsky images were merged. (G′-I′) Enlarged images of bracketed regions. (G′′-I′′) Enlarged confocal images of bracketed regions. Gradation of Bar expression gradually apparent until mid third instar (H-I). Note that, in I-I′′, Bar-negative tarsal segment 3 is physically separated from adjacent segments. (J-P) Early pupal legs (5 hours APF) stained for BarH1, ta5- lacZ or Ap. Green (J-L,N,O), BarH1; red (J,K) and green (M), ta5-lacZ; red (M,N,P), Ap. (M) Tarsal segment 4 and proximal edge of tarsal segment 5 specifically express Ap. Note that the strongest BarH1 expression occurs in tarsal segment 5. (Q) ta5-lacZ (green) and Ap (red) begin to be expressed just after the onset of central folding. (R) A late third instar disc stained for en-lacZ (brown) and BarH1 (black). (S,T) Staining for neu-lacZ (red) and Bar (green) indicates Bar− patches to be SOPs and/or their progenitors (arrows). For A-F,Q,R, anterior is left and dorsal is up. For other discs, distal is left and dorsal is up. Scale bar in F, 100 µm for A-Q, 70 µm for R, 50 µm for S,T.

1E,F,S,T). Staining for BarH1 and neu-lacZ (Bellen et al., type, see Fig. 1D). Together, these results may indicate that at 1989; Huang et al., 1991) showed that these correspond to least one of the five genes uncovered by Df(1)B263-20 is sensory organ precursors and/or their derivatives (Fig. 1S,T). essential for distal leg morphogenesis. To determine which cells require Df(1)B263-20 gene activity Functional requirements of Bar for distal tarsus for normal distal leg development, small Df(1)B263-20 clones formation were generated using the FRT/FLP method (Xu and Rubin, As a first step to elucidate Bar functions during leg 1993). Partial fusion of tarsal segments 2-5 were frequently development, we carried out gynandromorphic mosaic analysis observed (see Fig. 3P,Q). Central fold formation was also (Hotta and Benzer, 1976) using a larval-lethal deletion prevented in Df(1)B263-20 clones generated along the periphery Df(1)B263-20, which uncovers BarH1 and BarH2 along with of the early Bar ring (data not shown), consistent with the forked (f; Hoover et al., 1993; Ishimaru and Saigo, 1993), notion that both central folding and segmentation among tarsal Fimbrin (Fim; Ishimaru et al., unpublished data), and an segments 2-5 require the Df(1)B263-20 gene(s). In mosaic legs unknown gene, X2 (see Fig. 2N). In legs totally composed of normal in appearance (n=132), all mutant clones, except for a cells hemizygous for Df(1)B263-20, tarsal segments 2-5 were single case, in which a mutant bristle was situated at the fused together into a small bulb-like, non-segmental structure proximal tip of tarsal segment 3, were observed outside tarsal having neither claws nor pulvilli, whereas other leg segments segments 3-5 (Table 1), indicating that Df(1)B263-20 gene were normal (Fig. 2A,B). The first appreciable morphological activity in future tarsal segments 3-5, which are the presumed event in distal leg development is central folding along the derivatives of the early Bar ring, is essential for normal leg periphery of the Bar ring (see Fig. 1). As shown in Fig. 2K, no development. central folding occurred in Df(1)B263-20 leg discs; more Among five Df(1)B263-20 genes, f, Fim, BarH2 and X2 proximal folds (mf and pf) were normally formed (for the wild- appeared dispensable for distal leg development other than 772 T. Kojima, M. Sato and K. Saigo

Fig. 2. Leg phenotypes of Bar locus mutants and effects of BarH1 misexpression on leg morphology. 1-5, tarsal segments 1-5; cl, claws; ti, tibia; cf, central fold; mf, middle fold; pf, peripheral fold. (A) A distal part of the wild-type leg; (B) a gynandromorph mosaic leg consisting of Bar− (Df(1)B263-20) cells. Arrow, fusion of segments distal to tarsal segment 1. Neither claws nor pulvilli can be seen. (C,D) BM2/Df(1)B263-20 (C) and B55 (D) legs. Tarsal segments distal to tarsal segment 2 are fused. (E-J) Leg discs of BM2 (E,H) and B55 (F,G,I,J) stained for BarH1 (E-G) or BarH2 (H-J). In early third-instar-larval BM2 discs (E,H), BarH2 is almost normally expressed, but virtually no BarH1 ring is observed. Arrowheads, Keilin’s organ cells. In B55 discs, BarH1 is hardly expressed at both early (F) and late (G) third instar larval stages, while early BarH2 expression is almost normal (I); late circular BarH2 expression is significantly reduced (J). Note that the central folding occurred normally. (K) A mid third-instar disc of a gynandromorph mosaic larva, lacking BarH1 staining. No central fold formation occurs (compare (K) with Fig. 1D). (L,M) blk-GAL4/UAS-BarH1M13 discs stained for BarH1 (green). Confocal and Nomarsky images were merged. At mid third instar (L), ectopic fold formation occurs along the boundary between Bar-positive and Bar-negative tissues (arrowheads), while the formation of the authentic central fold is interrupted by Bar-misexpression (arrow). At late second instar (M), no folding occurs irrespective of BarH1 misexpression. (N) Physical map of the Bar locus and Bar mutant structures. The P insertion site in P058 is shown by a triangle. Distal inversion breakpoints in BM2 and pdf are indicated by vertical arrows. Filled boxes and thick arrows, transcriptional units. A stippled box, tarsal-segment-5-specific enhancer. Open boxes, breakpoints in B1, Df(1)B263-20, Df(1)BH2, BSY and B55. B1 is a duplication of the Bar locus (Lindsley and Zimm, 1992). Df(1)B263-20 is a deletion of B1 and lacks f, Fim, BarH2, BarH1 and X2. Df(1)BH2 is a deletion mutant lacking f, Fim and BarH2 (Higashijima et al., 1992b). BSY originates from B1 chromosome (Brosseau et al., 1958) and possess BarH1 but not other Df(1)B263-20 genes. (A-D) Distal is left; (E-M) anterior is left and dorsal is up. Scale bar in B: 50 µm for A-D, 80 µm for E-M.

bristle morphogenesis at least in the presence of BarH1. Bar deletion mutant uncovering BarH1, X2 and a late Bar Indeed, no defect in gross morphology of distal legs was enhancer (ta5) but not BarH2 (Fig. 2N). In B55 mutants, BarH1 observed when Df(1)B263-20 homozygous clones were expression is missing throughout development (Fig. 2F,G), while generated in flies carrying BSY, a Y chromosome with a Bar BarH2 expression was normal in early third instar larval stages locus fragment including BarH1 but not other Df(1)B263-20 but extensively reduced afterwards (Fig. 2I,J). As with pdf flies, genes (Fig. 2N, unpublished data). Consistently, no defect in tarsal segment 3 and more distal segments were fused together distal leg gross morphology was observed in Df(1)BH2, uncovering f, Fim and BarH2 (Fig. 2N, unpublished data; Higashijima et al., 1992b). Table 1. FRT/FLP mosaic analysis BarH1 may be functionally redundant to BarH2 as has been Segment No.* % shown in other developmental contexts (Hayashi et al., 1998; coxa 40 30 Sato et al., 1999). BM2 and pdf are inversion mutants with a trochanter 58 44 distal breakpoint between BarH1- and BarH2-coding femur 97 73 tibia 79 60 sequences (Fig. 2N; Tsubota et al., 1989). In these mutants, tarsal segment 1 40 30 early BarH1 expression was almost completely missing but tarsal segment 2 29 22 early BarH2 expression along with central folding occurred tarsal segment 3 1‡ 1 normally (Fig. 2E,H). We conclude that the genes essential for tarsal segment 4 0 0 distal leg morphogenesis are BarH1 and BarH2, which are tarsal segment 5 0 0 functionally redundant to each other. *The number of Bar− clone-possessing segments. A total of 132 legs In pdf and BM2/Df(1)B263-20 flies, tarsal segments 3-5 were normal in gross morphology were examined. partially fused with each other (Fig. 2C). B55 is a newly isolated ‡A y− bristle was observed at the proximal tip of tarsal segment 3. Leg segmentation by Bar homeobox genes 773

(Fig. 2D), while tarsal segment 2 and central fold were normally folding (Fig. 2M), or within the Bar ring and future pretarsus formed (Fig. 2D,F,I). The pdf and B55 was eliminated (Fig. 2L), suggesting the involvement of temporal and regional by BSY (Lindsley and Zimm, 1992; unpublished data). Based on factors other than Bar. these results, we conclude that late Bar function is involved in segmentation among tarsal segments 3-5. Requirements of Bar for specification of tarsal segments 3-5 Induction of folding by juxtaposition of Bar-positive Using mosaic analysis and misexpression, we examined and Bar-negative tissues whether Bar activity is required for the expression of markers Juxtaposition of Bar-positive and Bar-negative tissues may for tarsal segments 4 (ap) and 5 (ta5-lacZ). Neither Ap nor ta5- result in folding. To test this idea, BarH1 or BarH2 was lacZ expression was detected in Bar− clones (Fig. 3A-F). Bar misexpressed along the AP border using the UAS/GAL4 misexpression along the AP border caused ectopic expression system (Brand and Perrimon, 1993). blk-GAL4 or ptc-GAL4 of either ta5-lacZ or Ap or both in Bar-misexpressing regions was used as a GAL4 driver. As anticipated, no appreciable proximal to the Bar ring (Fig. 3G-I). Furthermore, Bar− clones difference in morphological changes were detected between within tarsal segments 4 and 5 were occasionally associated BarH1 and BarH2 misexpression. Fig. 2L shows that ectopic with campaniform sensilla (arrowhead in Fig. 3P), normally folding was formed along outer circumference of Bar- situated only at the dorsodistal tip of tarsal segment 3 (Fig. 3O). misexpressing region proximal to the Bar ring, while the Based on these observations, we conclude that Bar is essential formation of the authentic central fold was prevented in the for ap and ta5-lacZ (Bar) expression in distal tarsi and prevents regions where endogenous Bar-expressing cells and cells cells expressing Bar at later stages from adopting the tarsal solely expressing exogenous Bar were juxtaposed to each segment 3 fate. other. It would thus follow that central folding along the early ap-GAL4 is a GAL4 enhancer trap of the ap locus (Calleja Bar ring is an outcome of the juxtaposition of Bar-positive and et al., 1996). In ap-GAL4/UAS-Bar leg discs, not only did ta5- Bar-negative tissues. It should, however, be emphasized that no lacZ misexpression occur weakly in presumptive tarsal ectopic folding ever occurred either prior to the onset of central segment 4 (Fig. 3M,N), but also tarsal segment 4 occasionally

Fig. 3. Defects in tarsal segments 3-5 due to the absence or overexpression of Bar. 1-5, tarsal segments 1-5; pt, pretarsus. (A-F) Late third (A,B,D-F) or early pupal (C) leg discs having Bar− clones stained for BarH1 (A,C,D,E, green) and Ap (A-C, red) or ta5-lacZ (D,F, red). Focal planes of A,B and D-F are at tarsal segment 4 and tarsal segment 5, respectively. Ap and ta5-lacZ signals are abolished in Bar− clones (brackets). (G-I) A late-third-instar ptc-GAL4/UAS- BarH1M6 leg disc stained for ta5- lacZ (G,H, green) and Ap (G,I, red). Note that the central knob is slightly squeezed. Both ta5-lacZ and Ap misexpression are induced (arrowheads). (J-N) ap- GAL4/UAS-BarH1M13 leg discs. (J-L) An early pupal leg stained for BarH1 (K, green) and Ap (L, red). (J) Merged figure. Note that Bar expression levels in tarsal segment 4 are virtually identical to those in tarsal segment 5 (compare K with Fig. 1O). No appreciable Ap repression can be seen. (M,N) A sagittal section of a late third instar disc stained for ta5-lacZ (M,N, green) and Ap (M, red). Weak but significant ta5-lacZ expression is observed in tarsal segment 4 (arrowheads), suggesting partial fate change of tarsal segment 4. (O) A dorsal joint between wild- type tarsal segments 3 and 4. It is associated with a pair of campaniform sensilla (arrowheads). (P,Q) Legs possessing Bar− clones (bracketed regions). Ectopic joint (arrow) and campaniform sensillum (arrowhead) are formed in P, while partial fusion of tarsal segments 4 and 5 occurs in Q. (R) Ventral region of tarsal segments 4 and 5 in wild-type leg. The distalmost bristle of tarsal segment 5 (arrowhead) is colorless and thin, while that in tarsal segment 4 (arrow) is black and thick. (S,T) An ap-GAL4/UAS-BarH1M13 leg possessing tarsal segment 4/5 fusion. The bracketed region is enlarged in T. The black thick bristle at the distal edge of tarsal segment 4 (arrow) appeared transformed to that of tarsal segment 5 type, thin and less-pigmented. Arrowhead, authentic tarsal-segment-5 bristle. (A,B,D-I) Anterior is left and dorsal is up; (C,J-T) distal is left. Scale bar in A: 60 µm for A-F,H-N; 120 µm for G; 20 µm for O,P,R,T; 40 µm for Q,S. 774 T. Kojima, M. Sato and K. Saigo

Fig. 4. Requirements of Bar for antennal development. (A,B) Wild- type early (A) or late (B) third instar antennal discs stained for BarH1. Central fold is formed just outside the Bar ring (B, arrowhead). (C) A late third instar antennal disc of a gynandromorph mosaic larva lacking BarH1 signals. No central fold is formed. Arrow, Bolwig’s nerve stained with a neuron-specific antibody. (D) A wild-type antenna. ar, arista; b, basal cylinder. (E) A gynandromorph mosaic antenna consisting of Bar− cells. The arrowhead shows the absence of arista and basal cylinder. (A-C) Posterior is up and ventral is right. Scale bar in A: 50 µm for A-C; 30 µm for D,E.

showed partial transformation into tarsal segment 5. The ventralmost bristle situated at the distal tip of tarsal segment 4, normally thick and black (arrow in Fig. 3R), was frequently transformed into a thin and less-pigmented bristle (Fig. 3T) characteristic of tarsal segment 5 (arrowheads in Fig. 3R,T). Specific expression of ap or ap-GAL4 in future tarsal segment 4 reflects that subdivision of the early Bar ring has already occurred. Thus, the above findings may indicate that Bar misexpression in future tarsal segment 4 at later stages is still capable of causing future tarsal segment 4 cells to adopt tarsal is important for them to acquire and maintain the tarsal segment 5 fate at least partly, and hence, suggests that late segment 5 identity. It should, however, be noted that ap strong Bar expression in wild-type tarsal segment 5 primordia expression is not eliminated upon Bar misexpression (Fig.

Fig. 5. Antagonistic interactions between Bar and al and tarsal segment 5/pretarsus boundary formation. Leg (A,B,E- G,I-O,Q,R) or antennal (C,D,H) discs are stained for BarH1 (green) and Al (A-F,I-K, red), Fas II (O,Q,R, red) or with Rhodamine-phalloidin (M,N, red). (A-D) Wild-type discs. In leg discs, Bar and Al domains are overlapped each other in early third instar (A) but not in mid third instar (B). Insets, enlargements of boxed regions. In antenna discs, Bar and Al are initially expressed in the center (C) in that Al is expressed only within the Bar domain. Later, Bar expression but not Al expression is excluded from the center, so that Bar and Al domains are only slightly overlapped each other (D). (E,F) Leg discs having Bar− clones. The Al domain apparently invades into the Bar− clone (arrowhead) when observed in late third instar (F). No invasion occurs in early third instar (E). (G,H) al2/Df(2L)al discs. Patches of ectopic Bar expression are seen (arrowhead) in the leg disc at late third instar (G) and Bar expression in the center persists even at late third instar in the antennal disc (H), suggesting the partial repression of Bar expression by Al. (I,J) A blk-GAL4/UAS-BarH1M13 disc at mid third instar. Bar misexpression causes al repression (see arrowheads). (K,L) A ptc-GAL4/UAS-al3 disc at late third instar. Bar expression is not affected by al misexpression (arrowheads). (M-O) Wild-type late third instar discs. (M,N) Apical (M) and slightly basal (N) sections. Large arrows, position markers. Pretarsus cells (pt) other than rectangular border cells (bc) are tightly packed in the apical region, while the tarsal segment 5 (ta5) cells are loosely packed. (O) Fas II expression in border cells. The boxed region is enlarged in the inset. Fas II is concentrated along border-cell boundaries. (P) An illustration of a part of the tarsal segment 5/pretarsus boundary. (Q) Fas II expression is interrupted by a Bar− clone (arrow). (R) A ptc-GAL4/UAS-BarH1M6 disc. Ectopic Fas II expression is induced along the ectopic Bar domain (arrowhead), while normal Fas II expression is partially abolished by Bar misexpression (arrow). Anterior is left and dorsal is up. Scale bar in A: 60 µm for A-L; 18 µm for M,N; 35 µm for O,Q,R. Leg segmentation by Bar homeobox genes 775

Fig. 6. dac/Bar and Dll/Bar interactions. Late second (A), early third (B-D,F-H) or late third (E,I,J) instar leg discs are stained for BarH1(green) and Dac (A-C,F-H, red), Myc (D, red) or Dll (I,J, red). (A) Dac is expressed before the early Bar ring becomes discernible. Arrowheads, Keilin’s organ cells. (B) Early Bar expression occurs just inside the Dac domain. Arrowheads, Keilin’s organ cells. (C) Just before the onset of the central folding, region not expressing Dac appears. Confocal and Nomarsky images were merged. (D) In dac− clones (arrowheads), Bar expression is normal. (E) In the dac3 disc, Bar is derepressed in trochanter (arrowhead). (F) Dac misexpression is observed in a Bar− clone (arrowhead and inset). (G,H) Dac expression is repressed by Bar misexpression (arrowheads) in a ptc- GAL4/UAS-BarH1M6 disc. (I) In Dll− clones (arrowheads), Bar expression is abolished. (J) In Bar− clones (arrowhead), Dll is normally expressed. Anterior is left and dorsal is up except for C, distal is left and dorsal is up. Scale bar in A: 50 µm except for C, 100 µm.

3I,L,M), suggesting that a factor other than Bar is involved in Regionally exclusive expression of Bar and Al may be due repression of ap in tarsal segment 5. Together, these findings to mutually antagonistic interactions between Bar and Al. indicate that graded Bar expression at later stages may be When Al expression was examined in mid to late third instar essential for specification of tarsal segments 3-5. larval leg discs having Bar− clones, Al expression invaded a Ectopic incomplete segmental joints were frequently formed Bar− presumptive tarsus region (Fig. 5F), while Al expression in the vicinity of the boundary between Bar− clones and Bar- was considerably attenuated by Bar misexpression along the expressing tarsal segments 4-5 tissues (Fig. 3P). Tarsal anteroposterior compartment border in mid third instar discs segments 4 and 5 were partially fused with each other in ap- (Fig. 5I,J). Ectopic patches of Bar expression were frequently GAL4/UAS-Bar legs (Fig. 3S). Thus, the juxtaposition of observed in the presumptive pretarsus of hypomorphic al leg tissues expressing different levels of Bar may be essential for discs in late third instar (Fig. 5G). Bar derepression due to proper segmentation in distal tarsi. reduction in Al activity is more clearly observed in antennal discs; on a hypomorphic al mutant background, Bar was Requirements of Bar for distal antennal structure formation In Drosophila, antennae possess segmental structures homologous to those in legs and similarly differentiate through circular folding. Arista and basal cylinder, probably corresponding to pretarsus and distal tarsus (Postlethwait and Schneiderman, 1971), are derivatives of hth dac dac Dll Dll the central knob of antennal discs. After the onset of third instar, Bar expression occurred initially in a central region early Bar-ring early of the antennal disc and gradually became a ring similar to 3rd that observed in the leg disc (Fig. 4A,B). As with legs, instar dac dac central folding occurred just outside the Bar ring (Fig. 4B). Bar al In antennal discs lacking Bar activity, no central fold was formed (Fig. 4C); Bar− antennae frequently lost arista and basal cylinder (Fig. 4D,E). Thus, Bar is concluded to be dac dac essential not only for leg but also for antennal development. X Bar al Establishment of the tarsus/pretarsus boundary al is a homeobox gene expressed at the center of leg and folding antennal discs from early third instar onwards (Campbell et al., 1993; Schneitz et al., 1993). Initially, the Al ap Z ap late dac Y expression domain and early Bar ring overlapped slightly 3rd instar Bar Bar Bar al Bar al al in leg discs (Fig. 5A). Al/Bar overlapping could be more FasII FasII clearly seen in early antennal discs (Fig. 5C). Up to 90 hours AEL, the central part of the leg disc was strictly tarsal tarsal tarsal tarsal tarsal segment 1 segment 2 segment 3 segment 4 segment 5 pretarsus pretarsus divided into two regions, Bar-positive/Al-negative and Bar- border ()cells negative/Al-positive circular domains (Fig. 5B). In antennal discs, such discrimination in Bar/Al expression Fig. 7. Distal leg development is schematically summarized. Black, may be incomplete (Fig. 5D). expressed genes. Gray, repressed genes. For detail, see text. 776 T. Kojima, M. Sato and K. Saigo expressed in the centralmost region even at late third instar domains through hth, dac and Dll expression (Abu-Shaar and larval stages (Fig. 5H). That no appreciable repression of Bar Mann, 1998). At early third instar, circular expression of Bar was detected when al was misexpressed by ptc-GAL4/UAS-al and al begins within the Dll domain; Dll is required for the (Fig. 5K,L) may indicate the involvement of factors other than expression of Bar (Fig. 6I and unpublished data) and al Al in Bar repression in presumptive pretarsus. In addition, the (Campbell and Tomlinson, 1998). Future tarsal segment 2 lack of Al invasion into Bar− clones in leg discs at early third may be generated in the distalmost region of the Dac ring instar larval stages (Fig. 5E) may indicate that Bar is possibly through repression of dac expression by an unknown dispensable for repressing al when their expression is initiated. factor, X (Fig. 7), since Dac is expressed in the region At late third instar, cells in the distalmost region of leg discs immediately proximal to the early Bar ring (future tarsal are densely packed at the apical surface and distinguishable segments 3-5) at early stages (Fig. 6B) but not in tarsal from surrounding loosely packed cells (Condic et al., 1991; segment 2 at later stages (Abu-Shaar and Mann, 1998; Lecuit Fig. 5M). Double-staining with rhodamine-phalloidin and anti- and Cohen, 1997). BarH1 antibody revealed that the former corresponds to Bar- Expression of molecular markers for tarsal segments 5 (ta5- negative pretarsus cells, and the latter, Bar-positive tarsus cells lacZ) and 4 (ap) becomes apparent within the Bar ring just after (Fig. 5M,N). Proximalmost pretarsus cells (border cells) were the onset of central folding but before distal tarsus segmentation frequently rectangular in apical shape (Fig. 5M). Staining for (Fig. 1Q). Cells in the proximalmost region of the early Bar ring Fasciclin II (Fas II; Grenningloh et al., 1991) showed that may also be committed to become tarsal segment 3 at this stage. border cells prominently express Fas II at late third instar Bar expression within the early Bar ring is nearly homogeneous (Fig. 5O). Fas II expression was interrupted by Bar− clones (Fig. 1B) and thus the initial subdivision of this ring into future (Fig. 5Q). Fas II misexpression was induced along Bar- tarsal segments 3-5 may require factor(s) other than Bar. ss is misexpressing presumptive pretarsus, while endogenous Fas II expressed transiently in the future tarsus region in late second expression was repressed (Fig. 5R). Thus, Bar would to early third instar and ss mutant legs lack tarsal segments 2- upregulate and downregulate Fas II expression in Bar-negative 4 but not 5 (Duncan et al., 1998). ss may thus be responsible border cells and Bar-positive non-border cells, respectively. for differential between future tarsal segments We, thus, conclude that Bar is essential for the establishment 4 and 5. Repression of Bar expression in future tarsal segment of the boundary between tarsal segment 5 and pretarsus. 3 may be due to a putative Bar repressor, Y (Fig. 7). At later stages, Bar expression is strong in tarsal segment 5 Interactions between Bar and dac or Dll (Fig. 7, deep green), moderate in tarsal segment 4 (light green) Circular Dac expression appeared in second-instar leg discs and absent from tarsal segment 3. Genetic and morphological before Bar ring appearance (Fig. 6A). This early Dac-ring was analyses (Table 1; Fig. 3) strongly suggest that Bar upregulates associated interiorly with Bar-positive Keilin’s organ cells, ap and/or its own expression in tarsal segments 4 and 5. Since which are situated along the interior circumference of or within (1) only partial transformation of tarsal segment 4 into 5 occurs the early Bar ring (see Figs 1A, 6A,B). Although they were upon Bar overexpression in tarsal segment 4 (Fig. 3T) and (2) separated from each other by a Bar-negative, Dac-negative Bar misexpression fails to repress Ap expression (Fig. 3G-I), region just before the onset of central fold formation (Fig. 6C), an unknown factor (Z) is likely involved in ap repression in Dac and Bar rings were immediate neighbours at earlier stages future tarsal segment 5 (Fig. 7). (Fig. 6B). Dac expression was derepressed in Bar− clones Duncan et al. (1998) suggest that tarsal development takes observed in early third instar (Fig. 6F), while repressed by Bar place in two steps: establishment of a uniform tarsal region misexpression (Fig. 6G,H), indicating that Bar is essential for followed by subdivision of this ring into segments. Our results distal restriction of Dac expression. Since early Bar expression indicate that there are several more intermediate steps in this normally occurred in dac− clones (Fig. 6D), dac appears process. Abu-Shaar and Mann (1998) propose three phases of dispensable for proximal restriction of the early Bar ring. leg-disc subdomain formation during early development Interestingly, Bar misexpression occurred in future trochanter probably prior to the onset of Bar expression. Thus, leg in dac− mutants (Fig. 6E), indicating that Dac represses Bar in segmentation requires repeated subdivision of leg epithelium future trochanter. along the proximodistal axis with the result that smaller region- Dll is expressed from the beginning of leg development specific domains are generated from larger (Abu-Shaar and Mann, 1998). The Dll domain includes all ones in all instances of subdivision. Bar-expressing cells (see Fig. 6I,J). Bar expression was abolished in Dll− clones (Fig. 6I), while Dll continued to be Genetic interactions of Bar with al expressed in Bar− clones (Fig. 6J). Bar expression thus appears Bar and Al expression begins essentially at the same time at to require Dll activity but Dll does not require Bar. early third instar. Initially, Al expression partially overlaps Bar expression (Figs 5A,C, 7, yellow), and no invasion of Al into Bar− clones was found when mosaic clones were observed at DISCUSSION this stage (Fig. 5E). However, at slightly later stages, Al and Bar expression became mutually exclusive (Fig. 5B,D) and Al Mechanism of distal leg development invaded into Bar− clones (Fig. 5F). It may thus follow that al Distal leg segmentation is a multiple-step process involving expression is initiated Bar independently and, after a while, various aspects of development. Our results are summarized in Bar protein accumulated to some extent begins to repress al Fig. 7. Most events of distal leg segmentation occur during expression. al may also regulate Bar expression in a similar third instar larval stages. fashion. Indeed, Bar misexpression occasionally occurred in By early third instar, the leg disc has been divided into four leg and antennal discs of al hypomorphic mutants (Fig. 5G,H), Leg segmentation by Bar homeobox genes 777 although Bar expression was not repressed by al misexpression clone. We are also grateful to Y. Hotta, G. Mardon, I. Guerrero, R. (Fig. 5K,L). The failure of Bar repression by al may suggest Ueda, FlyView and Bloomington Drosophila stock center for fly the involvement of other pretarsus gene(s) that function strains. This work was supported in part by grants from the Ministry cooperatively with al. We actually identified two new genes of Education, Science Culture and Sports of Japan to T. K. and K. S. that function in the pretarsus and show mutant phenotypes similar to al (T. Tsuji, T. K. and K. S., unpublished data). Bar misexpression experiments (Fig. 3G-I) showed REFERENCES pretarsus to be the region where ta5-lacZ expression is very difficult. 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